RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair

Summary Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.


INTRODUCTION
2][3][4][5][6][7][8] BRCA2 is a recombination mediator suppressing hereditary breast and ovarian cancer. 9To mediate HR, BRCA2 associates with RAD51 via two regions: the BRC repeats 10,11 and exon 27 (Ex27). 12,135][16][17][18] Mechanistically, BRC4 or BRC 1-8 stimulates RAD51 binding to ssDNA 19 and BRC repeats (BRC1-4) block RAD51 ATP hydrolysis permitting the accumulation of an ATP-bound nucleoprotein filament. 20Ex27 binds to an interface created by two adjacent RAD51 monomers 21,22 to protect and restart RFs. 23Cells expressing BRCA2 deleted for Ex27 exhibit hypersensitivity to g-radiation, premature replicative senescence, chromosomal instability, increased stalled RF, and reduced survival due to early cancer onset. 2,24,25ere, we elaborate on the role of the RAD51-Ex27 interaction.We show that Ex27 does not influence ATP hydrolysis but stabilizes RAD51 filaments from dissociation in the presence of a GST-BRC3 trap.We identify a mutation of serine 181 (S181P) that selectively inhibits the interaction between RAD51 and Ex27.In contrast to the well-described RAD51 K133R mutant, RAD51 S181P is able to hydrolyze ATP but Ex27 cannot stabilize the filament.Importantly, the RAD51-Ex27 association is dispensable for repairing DSBs but is required for RAD51-mediated protection of stalled RFs to preserve chromosomal integrity.

RAD51 S181P reduces the interaction with Ex27
The mutagenic yeast-two hybrid protocol 26 was used to isolate a mutation that diminished RAD51's interaction with Ex27 but maintained interactions with RAD54, BRC3, and itself.Upon screening 2,500 colonies, a mutant was isolated that failed to grow with Ex27 but grew with the other partners (Figure 1A) and sequencing revealed a serine to proline change in residue 181 (S181P or SP).RAD51 S181P interaction with BRC3 but not with Ex27 was validated by co-immunoprecipitation (Figure S1A) and further confirmed in an in vitro pull-down assay using purified proteins (Figure 1B).To determine the requirement of specific change from serine 181 to proline for interaction with Ex27, we generated and tested recombinant RAD51 S181A and S181R mutants.By using biolayer interferometry (BLI) and in vitro pull-down assays, we observed that both S181A and S181R were capable of associating with Ex27 (Figures S1B and S1C), suggesting that loss of interaction can be attributed to the proline residue.

Ex27 peptide fails to bind and stabilize RAD51 SP filaments in vitro
Previous studies have proposed that Ex27-RAD51 interaction is important for protecting stalled RFs against MRE11-mediated degradation but is dispensable for HR-dependent repair of DSBs. 6For comparison purposes, we purified and examined DSB repair-deficient RAD51 K133R (KR) mutant that is Ex27-interaction proficient (Figure 1B) but, in contrast to RAD51 SP, deficient in ATPase activity 27 (Figure 1C).A double-mutant, RAD51 K133R/S181P (KR/SP) was also generated, which displayed deficiency in both Ex27-interaction and ATPase activity (Figures 1B and 1C).
Next, we analyzed the biochemical properties of SP both individually and in combination with Ex27 and compared them to KR and KR/SP.Using a D-loop assay in the presence of magnesium ions, we show that RAD51 wildtype (WT) and SP displayed comparable levels of D-loops (Figure S2A).Conversely, KR and KR/SP mutants showed significantly higher D-loop yields under the same conditions (Figure S2A), which correlates with deficient ATPase activity and formation of more stable ATP-bound filaments.In the presence of calcium ion, which induced comparable levels of D-loop for all tested proteins, the addition of Ex27 hindered D-loop formation for WT and KR, while SP and KR/SP mutants remained resistant to this inhibition (Figures 2A and 2B).BRC repeats were shown to inhibit RAD51 ATPase activity; 14 therefore, we tested the influenced of ATPase activity on RAD51-ssDNA complexes.Unlike GST-BRC3, Ex27 peptide did not affect RAD51 ATPase activity, even at a 10-fold excess over RAD51 WT (Figure 2C).
To examine whether RAD51 SP filaments fail to be protected against BRC destabilization in the presence of Ex27, pre-formed RAD51 filaments on fluorescently labeled ssDNA were exposed to Ex27 peptide and subsequently challenged with GST-BRC3 to monitor RAD51 dissociation in a gel shift assay.The binding of Ex27 to RAD51-ssDNA filaments was evident as an additional super-shift of migrating RAD51-ssDNA complexes for WT and KR but not for SP and KR/SP (Figure S2B).Upon challenge by GST-BRC3, free ssDNA was released from RAD51-ssDNA complexes, indicating complex disassembly (Figure S2B).In the presence of Ex27, RAD51 WT, and KR-ssDNA filaments are almost completely resistant to GST-BRC3, suggesting stabilization by Ex27 (Figure 2D).In contrast, SP and KR/SP filaments were not protected (Figures 2D and  S2B), further supporting the loss of Ex27 interaction.These data indicate that while Ex27 cannot inhibit the ATPase activity of RAD51 WT, it does reduce D-loop formation, possibly via binding and stabilization of RAD51-ssDNA filament.
Our previous methods are limited to monitoring protein-DNA binding at the steady state.We thus employed pre-steady state analysis using stopped-flow to address potential alterations in RAD51 filament assembly, specifically focusing on RAD51 SP filaments and the effect of Ex27 (Figures S2C and S2D).This technique allowed us to monitor the kinetics of protein-ssDNA complex assembly in real-time by mixing RAD51 with a 5 0 -Cy3 fluorescently labeled (dT) 79 oligonucleotide (Cy3-79-mer). 28The binding of RAD51 to 5 0 -Cy3-(dT) 79 leads to an increase in Cy3 fluorescence signal due to Cy3 isomerization, known as protein-induced fluorescence enhancement (PIFE).The relative fluorescence change over time serves as a proxy of protein-DNA complex formation.The assembly of RAD51 filaments involves complex binding mechanism, 29 and we observed a similar mechanism for the SP protein but noted slower kinetics of assembly at suboptimal concentrations (Figure S2E).In the presence of Ex27, we observed changes in the kinetics of WT filament assembly by increasing overall binding amplitude and decreasing binding rate (Figure S2C).In contrast, Ex27 had no effect on the SP mutant (Figure S2D), confirming the loss of interaction.
Furthermore, a BLI-based assay was used to monitor RAD51 filament disassembly. 30RAD51 filaments were assembled on biotin-ssDNA immobilized on a streptavidin biosensor tip, and their dissociation by BRC4 peptide was observed over time.The data from the dissociation phase were normalized to the starting point, and the amplitude of the change was plotted as a function of time.While the BRC4 peptide strongly induced the disassembly of both WT and SP filaments, the addition of Ex27 only suppressed RAD51 dissociation from DNA for the WT protein (Figures 2E, S2F, and S2G).Accordingly, no association of Ex27 with SP filament was detected by BLI (Figure 2F).

RAD51 S181P cells are proficient in HR-mediated DSB repair
To assess the cellular phenotype of human RAD51 WT and SP, a knockout-knockin protocol in mouse embryonic stem (ES) cells (AB2.2) was used to introduce corresponding cDNAs adjacent to the mouse RAD51 promoter while leaving the remaining mouse allele intact. 31RAD51 KR and KR/SP cDNAs were also included.An essential component of this protocol is the miniHPRT gene, which can be selected for or against expression using HAT (hypoxanthine, aminopterin, thymidine) and 6-thioguanine (TG), respectively 32,33 (Figure S3A).The genotypes are described as a mouse: AB2.2 RAD51 WT/+ or AB2.2 RAD51 (KR, SP, KR/SP)/+ and will be referred to as WT, KR, SP, and KR/SP for this paper.In contrast to previously reported reduced clonogenicity of KR mutant, 2 the SP had no effect compared to WT (data not shown).
A dose-response curve was recorded for camptothecin (CPT, type 1 topoisomerase inhibitor), olaparib (OLA, PARP1 inhibitor), and X-rays to assess the response to clastogenic agents.CPT and OLA induce damage associated with replication, 34,35 while X-rays cause mainly replication-independent DSBs plus a variety of replication-associated damage, including modified bases, single-strand breaks, and protein-DNA crosslinks. 36Treatment with CPT and OLA was initiated upon the cell-seeding and continued throughout the experiment. 37Cell number was counted five days later.Unlike KR and KR/SP cells, SP cells were not hypersensitive to CPT and OLA (Figure 3A, left & middle panels); however, none of these mutants displayed differential sensitization to X-rays (Figure 3A, right panel), possibly reflecting replication requirement for CPT and OLA-induced damage.
The proficiency of DSB repair via HR was evaluated using the DR-GFP assay in U2OS cells overexpressing RAD51 variants.KR and KR/SP cells showed a significant drop in number of GFP-positive cells (Figure 3B, left panel), consistently with a previously reported dominant-negative effect of KR expression on HR efficiency. 27In contrast, SP cells exhibited a similar number of GFP-positive cells compared to WT, indicating their comparable HR proficiency.To assess the efficiency of other DSB repair pathways, the EJ5-GFP and SA-GFP assays were performed in U2OS cells.The EJ5-GFP assay measures non-homologous end-joining (NHEJ), a pathway that rejoins DNA ends without using a homologous template.In contrast, the SA-GFP assay evaluated single-strand annealing (SSA), which joins complementary ends when repeats flank the DSB. 38,39The NHEJ levels were comparable in all cell lines (Figure 3B, middle panel) while the level of SSA was significantly elevated in KR and KR/SP cells, pointing to a compensatory mechanism in these HR-deficient cells (Figure 3B, right panel).These observations cannot be explained by differential RAD51 expression levels as shown by western blot (Figure 3B, bottom).Our data suggest that the RAD51-Ex27 (E) Ex27 failed to protect SP filaments against dissociation by BRC4 peptide monitored by BLI.WT (blue) and SP (green) filaments were pre-formed on immobilized ssDNA (oligo-dT, 43-mer), and RAD51 dissociation was followed in the presence of BRC4 and Ex27.Off-rates of RAD51 dissociations are plotted, for dissociation curves in Figures S2F and S2G.(F) Binding of Ex27 to RAD51-ssDNA filaments using BLI.RAD51 WT (blue) and SP (green) filaments were formed as aforementioned and the binding of Ex27 was detected in real time as an increase in optical thickness.
interaction is dispensable for DSB repair via HR, while RAD51 ATPase activity is critical.Accordingly, WT and SP cells were proficient in ATPase activity, while KR and KR/SP were not (Figure 1C).

RAD51 SP forms foci in cells but fails to alleviate replication stress
RAD51 WT and SP foci were visualized with green fluorescence by expressing RAD51 cDNA conjugated to enhanced green fluorescent protein (eGFP).WT mouse RAD51 was deleted in cells with eGFP-WT or eGFP-SP (Figure S3A) by using CRISPR/Cas9 (Figure S3B).In this manuscript, we refer to the genotypes as eGFP-WT or eGFP-SP for simplicity.The number of cells with R10 foci was comparable between unexposed eGFP-WT and eGFP-SP cells (Figures 4A and S4).Upon exposure to CPT (1 m M, 3 h) or X-rays (1 Gy, 3 h release), the number of cells with R10 foci increased similarly for eGFP-WT and eGFP-SP (Figures 4A and S4).
To test the role of RAD51 mutants in maintaining RFs, we performed dose-response curves to three agents that influence replication: hydroxyurea (HU, a ribonucleotide reductase inhibitor), aphidicolin (APH, an inhibitor of DNA polymerase a, d, and ε) and VE-821 (an ATR inhibitor).No significant differences were observed in response to these agents (Figures S5A-S5C).These data are consistent with the mild sensitivity of RAD51 knock-outs to HU and ATRi, as compared to olaparib, reported in pooled CRISPR screens. 41Similarly, BRCA2 loss results only in subtle sensitization to HU compared to strong sensitization to olaparib. 6Therefore, even if some of the RAD51 mutants confer slight sensitivity to replication poisons in our system, where the RAD51 WT allele is present, these more-subtle effects may not be detectable in cell survival assays.However, HR is known to process stalled RFs after exposure to a mild dose of HU that does not cause breaks. 42To quantify stalled and restarted RFs by DNA fiber assay, cells were treated with IdU for 20 min followed by exposure to 0.5 mM HU for 1.5 h, and then CldU was added for 20 min.Compared to WT, all RAD51 mutants exhibited increased RF stalling without HU treatment and after HU treatment.Compared to WT, HU treatment increased RF stalls for all mutants (Figure 4B).In addition, all mutants also showed decrease in fork restart compared to WT (Figure 4B), indicating possible defect in fork protection and/or reversal.Stalled RFs are known to be susceptible to nucleolytic degradation by MRE11 nuclease in the absence of stable RAD51-ssDNA interaction. 6To investigate nascent strand protection, the CldU/IdU ratio was measured in cells treated with IdU for 30 min, followed by CldU for 30 min, and then 4 mM HU for 5 h.Compared to WT, the RAD51 mutants showed insignificant degradation without treatment (NT).Yet, all mutants exhibited nascent strand degradation after HU exposure compared to WT (Figure 4C).RF speed was evaluated by observing the length of the CldU fibers.Without HU treatment, CldU tract length showed no difference between WT an SP, yet longer fibers were seen with KR and KR/SP suggesting faster replication (Figure 4D).With HU treatment, the length of the CldU fibers were the same for WT, KR, and SP; however, the KR/SP were shorter suggesting reduced speed (Figure 4D), possibly due to a combination of deficient fork reversal and subsequent fork degradation (Figure 4C). 43

RAD51 SP causes a different array of chromosomal aberrations than KR
To investigate the potential differences in chromosomal alterations resulting from the differential requirements of ATPase activity and Ex27 interaction, we assessed chromosome stability using two-color fluorescence in situ hybridization (FISH) on metaphase spreads (MPSs). 44We scored various chromosomal aberrations, including Robertsonian translocations (RbT), extra pericentromeres and telomeres (EPTs), isochromatid breaks (ICBs), chromatid breaks (CBs), and radials (Figure 5A). 2 RbTs occur when two chromosomes join at the centromere, with or without telomeres, and could arise due to defective replication or chromosome fusion. 45EPTs are duplications resulting from faulty replication or imprecise joining of multiple DSBs and have been described in cells expressing RAD51 K133A, 2 a mutation that inhibits ATP binding.ICBs suggest failed strand exchange intermediates that break both chromatids, while CBs represent one-ended breaks at collapsed RFs.Radials are chromosomal structures formed by the fusion of multiple chromosomes and occur in cells derived from Fanconi anemia patients exposed to crosslinking agents. 46KR, SP, and KR/SP exhibited a 17-, 24-, and 56-fold increase, respectively, in spontaneous chromosomal abnormalities when compared to WT (Figure 5B; Table S1).KR and KR/SP displayed mostly ICBs and ICB/EPT, respectively, consistent with defective DSB repair (Figure 5B; Table S1).SP predominantly showed RbTs without central telomeres, indicating defective replication (Figure 5B; Table S1).Chromosome painting was performed on SP MPSs to further investigate SP cells, revealing two RbTs involving duplication of chromosome 8 with chromosome 8 disomy (Figure S6A).
The effect of dose-dependent genotoxic lesions on chromosomal instability was also analyzed.Cells were exposed to physiologically comparable doses of genotoxins (HU, CPT, and OLA) to ensure that differences in chromosomal instability between genotypes were not related to the dose of the genotoxin (Figures S6B-S6D).After exposure to genotoxins, KR and KR/SP cells exhibited increased levels of chromosomal defects, notably radials (Figure 5B; Table S1), which could result from elevated levels of NHEJ or SSA (Figure 2D).However, SP cells did not (D) Track length taken from the experiment described in F and converted into kb using conversion factor 1 mm = 2.59 kb. 40how a significant increase in radials except at a high dose of HU (Figure 5B; Table S1).Furthermore, a miniHPRT loss-of-function (LOF) assay was performed to assess the mutation rate in RAD51 variants by measuring cell survival in TG. 31 KR, SP, and KR/SP exhibited a spontaneous increase of 82-, 22-, and 3-fold, respectively, in TG-resistant colonies compared to WT (Figure 5C).Interestingly, the KR/SP mutant displays a lower mutation level than either KR or SP, suggesting that serine 181 promotes mutagenesis in KR cells, while lysine 133 likely plays a similar, but less prominent role in SP cells.These data support separate function for KR and SP mutants and may reflect use of alternative pathways for repair/mutagenesis.The overall decrease of mutagenesis in the double mutant may be a result of combined loss of both alternative pathways, where most of the damage remains unrepaired and cells mostly die.

DISCUSSION
Using the yeast two-hybrid screen, we isolated the S181P mutation in RAD51 that is defective in its interaction with BRCA2 exon 27 while retaining its ability to interact with the BRCA2 BRC3 motif and other RAD51-interacting proteins.During the revision process of this manuscript, a cryo-electron microscopy structure of BRCA2 exon27 with RAD51 was published. 47This study revealed an acidic patch, containing the RAD51 S181, mediating the interaction with both Ex27 and BRC4.Interestingly, while mutations of the acidic residues (D184, D187) attenuated binding of both Ex27 and BRC4, RAD51 SP mutant is selectively deficient for the interaction with Ex27.The introduction of proline residue is likely responsible for the loss of interaction since exchange of S181 for alanine, arginine, or cysteine did not compromise Ex27 binding (Figure S1; 47 ).
Biochemical characterization revealed that the SP mutant maintained ATPase activity, in contrast to KR and KR/SP.Interestingly, Ex27 did not affect the ATPase activity of RAD51-ssDNA complexes but exerted an inhibitory effect on D-loop formation.Previously, Ex27 was shown to stabilize RAD51-ssDNA filaments against BRC3-mediated disassembly. 21,22GST-BRC3 acts as a ''RAD51 trap,'' binding to dissociated RAD51 monomers and preventing their re-binding to ssDNA. 20Our findings show that Ex27 stabilizes RAD51 WT-and KR-ssDNA filaments against dissociation in the presence of GST-BRC3, yet it inhibits their ability to form D-loops.In contrast, SP and KR/SP mutants are dissociated by BRC3 but are not inhibited in forming D-loops, suggesting role of RAD51 turnover during this process.Consistent with these observations, our cellular data indicate that the interaction between Ex27 and RAD51 has a DSB repair-independent function, since SP, unlike KR, did not display a deficiency in HR-mediated DSB repair.
shown that in contrast to fork protection, its reversal depends on RAD51's ability to catalyze strand invasion. 43This points to a more specific role of Ex27-dependent regulation of RAD51 filament in controlling DNA accessibility during the processing of stalled forks.Notably, the RAD51 SP mutant differs from the human RAD51-II3A mutant that protects against MRE11 but not against DNA2. 49This phenotype is reminiscent of RAD51 T131P mutant, which forms less-stable filaments in vitro and results in the accumulation of pRPA on RFs but does not exhibit visible HR deficiency in the DR-GFP assay in cells. 50,51ficiency in RAD51 Ex27-interaction or its ATPase activity results in different chromosomal abnormality spectrum Compared to WT, all RAD51 mutant cells exhibited increased levels of spontaneous chromosomal abnormalities.KR cells predominantly exhibited isochromatid breaks while KR/SP cells showed a combination of isochromatid breaks and ETPs.These data are consistent with KR and KR/SP being defective in both HR-mediated DSB repair and RF maintenance.On the other hand, the most common aberrations observed in SP cells were RbTs.These RbTs did not have central telomers, and chromosome painting revealed a duplication of chromosome 8 with chromosome 8 disomy, suggesting a replication defect at the centromere.It has been reported that HR machinery, including RAD51, is recruited to breaks during G 1 and HR deficiency causes centromeric instability and chromosomal translocations. 52We speculate that the centromeric instability observed in SP cells arises as a consequence of RAD51-mediated processing of stalled RFs in peri-centromeres and centromeres.The ability of SP cells to repair breaks via HR and alternative pathways (NHEJ, SSA) may facilitate the specific formation of RbTs.Our data thus describe novel separation of function mutant that might help dissect the multifaceted role of RAD51 in DSB repair and processing of stalled RFs and explain some of disease-associated mutations in RAD51. 53

Limitations to the study
This study is limited to the functional characterization of RAD51 KR and SP in mouse ES and U2OS cells, but no organismal data are presented here (a description of the RAD51 SP/SP will be submitted in the near future).Another limitation is the lack of analysis of the functional integration of RAD51 with other aspects of RF maintenance.For example, the impact of KR and SP mutations on DNA damage tolerance (DDT) remains unexplored.Previously we showed that RAD51 and BRCA2 were responsible for the fusion of identical inverted repeats, while DDT was responsible for the fusion of mismatched inverted repeats. 54Although we have previously shown that RAD51 K133A mutation relies upon TREX2 function to induce nearly all the mutations in ES cells, 31 the effect of KR and SP mutants on these fusion events remains unknown.Further investigation of these mutants may provide valuable insight into distinguishing between strand exchange and RF protection and shed light on their utilization by alternative pathways.
To measure expression level of I-SceI and exogeneous RAD51, U2OS cells were seeded 3x 10 5 cells/well in 6-well plate.Next day cells were co-transfected with the I-SceI expression vector (pCBASce) and 87.5 ng of the RAD51 expression vector.Seventy-two hours later cells were harvested and analyzed by Western blotting.Membranes were probed with antibodies against human RAD51 (Bio Academia 70-002), HA-tag to detect I-SceI (Abcam ab9110) and Actin as a loading control (Abcam ab184220).

Analysis of replication forks
DNA fiber analysis was used to measure RF restart/stalling and nascent strand degradation as described. 31Briefly, for replication fork dynamics, the cells were pulsed with 25 mM IdU for 20 min, treated with 0.5 mM HU for 1.5 hr and pulsed with 250 mM CldU for 20 min.To prepare DNA fiber spreads, the cells were dropped onto superfrost microscope slides (Thermo scientific) and dried for 6 min.Then, the cells were lysed with spreading buffer (200 mM Tris-HCl pH 7.4, 50 mM EDTA, 0.5% SDS) and incubated for 2 min.The slides were tilted at 15 , and the DNA fibers were spread slowly down to the end of slides.The fibers were fixed in methanol/acetic acid (3:1) at RT for 10 min and air-dried.The fibers were washed with ddH 2 O, rinsed with fresh 2.5 M HCl for 2 min, and denatured in fresh 2.5 M HCl for 1.25 hr.They were incubated in blocking buffer (1% BSA, 0.1% Tween 20 in PBS pH 7.4) for 0.5-1 hr and followed by incubation with rat a-BrdU (1:650) and mouse a-BrdU (1:650) in blocking buffer for 1 hr.The fibers were rinsed and fixed in 4% paraformaldehyde for 10 min.Then, they were incubated with secondary antibodies (anti-mouse AlexaFluor 488 and anti-rat Alexa Fluor 555, 1:500) for 1.5 hr and were mounted with Fluoroshield.The DNA fibers were captured with Axio Imager A2 at 63x magnification (Zeiss) and analyzed using Zen 2.3pro software (Zeiss).For nascent strand degradation, the cells were pulsed with 25 mM IdU for 30 min, pulsed with 250 mM CldU for 30 min.The cells were treated with 4 mM HU for 5 hr.The next procedures were followed as above.At least 1000 fibers and 129 fibers were counted and analyzed for replication fork dynamics and nascent strand degradation, respectively.

Immunoprecipitation of RAD51 variants
The indicated plasmids were co-transfected using Lipofectamineä 3000 Transfection Reagent (invitrogen) according to the manufacturer's instructions.After 6 hr, transfection reagents were removed and changed to fresh medium.After 48 h, HEK293T cells were harvested for further experiments.Cells were lysed with buffer X (100mM Tris-HCl (pH 8.5), 250 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) and supplemented with the complete protease inhibitors cocktail (Roche) and PhosSTOP (Roche) for 1 hr at 4 C. Cell lysates were sonicated and centrifuged at 13000 rpm for 15 min at 4 C. Then cell lysates were incubated with Streptactin Sepharoseâ High Performance (GE Healthcare) for 1 hr at 4 C with constant rotation.After washing three times using buffer X, Streptactin-bound proteins were eluted by boiling samples in sample buffer (GenDEPOT), followed by immunoblotting with indicated antibodies.

Figure 1 .
Figure 1.RAD51 S181P (SP) is selectively deficient for the interaction with BRCA2 Ex27 (A) Yeast-two hybrid showing that SP associates with RAD54, RAD51, and BRC3 but not with Ex27.(B) In vitro pull-down showing SP interaction with RAD51 and BRC3 but not with Ex27.Gels stained with Coomassie Blue.I = input, F = flow-through, B = bound fractions.(C) The ATPase activity of RAD51 WT, KR, SP, and KR/SP in the absence (dotted lines) or presence of ssDNA [(dT)63] (solid lines).

Figure 2 .
Figure 2. RAD51 SP filaments were proficient for D-loop formation, but not Ex27 stabilization (A) Increasing amount of Ex27 inhibited D-loops for WT and KR, but not SP and KR/SP.Assay was performed in the presence of calcium and ATP to obtain similar levels of basal D-loop between all mutants.(B) Quantification of gels shown in panel A (Mean G SD, n = 3).(C) Unlike GST-BRC3 (7.5 mM), Ex27 failed to inhibit the ATPase activity of WT (Mean G SD, n = 3).(D) Ex27 peptide (7.5 mM) failed to stabilize SP and KR/SP protein-DNA complexes from BRC3-GST mediated disassembly (Mean G SD, n = 3).(E)Ex27 failed to protect SP filaments against dissociation by BRC4 peptide monitored by BLI.WT (blue) and SP (green) filaments were pre-formed on immobilized ssDNA (oligo-dT, 43-mer), and RAD51 dissociation was followed in the presence of BRC4 and Ex27.Off-rates of RAD51 dissociations are plotted, for dissociation curves in FiguresS2F and S2G.(F) Binding of Ex27 to RAD51-ssDNA filaments using BLI.RAD51 WT (blue) and SP (green) filaments were formed as aforementioned and the binding of Ex27 was detected in real time as an increase in optical thickness.

Figure 4 .
Figure 4. Analysis of replication fork dynamics in RAD51 mutant-expression cells (A) Graph summarizing the percentages of RAD51 foci for cells expressing eGFP-WT or eGFP-SP, either untreated (NT) or exposed to 1 mM CPT for 3 h or 1 Gy X-rays, followed by 1 h recovery.More than 184 cells were counted per each sample and the counting assessment was done blind.The graph is showing the percentage of cells without (no) or with foci (<10 or R 10) as a mean G SD (n = 3).Statistics (unpaired t-test) is indicated for the R 10 foci dataset: **p < 0.005, ***p < 0.0005.(B) Analysis for RF progression by DNA fiber assay.Left panel: Schematics of the assay, cells were exposed to IdU for 20 min, then a mild HU dose (0.5 mM, 1.5 h), followed by drug removal and treatment with CldU for 20 min.Three categories of phenotypes were scored: 1) restarted RF (green-red), 2) stalled RF (green), 3) new origin (red).At least 1000 fibers were analyzed for each sample.Statistics: a Chi-square with Yates' correction and Fisher's exact test.(C) Analysis for nascent strand degradation.Schematics of the assay: cells were treated with IdU (30 min) followed with CldU (30 min) and then HU (4 mM, 5 h).At least 129 fibers were observed for each sample.Short red lines demarcate the median for each sample, the long red line demarcates the median for WT no treatment (NT).Statistics: Kruskal-Wallis test with Dunn's multiple comparison.Not significant (NS), *p < 0.05, **p < 0.005, ***p < 0.0005.(D)Track length taken from the experiment described in F and converted into kb using conversion factor 1 mm = 2.59 kb.40